Human immunodeficiency virus regulatory protein Rev (regulator of viral expression) is translated from a monocistronic transcript produced early in the viral replication cycle. Rev binds to the cis-acting, highly structured viral RNA sequence Rev response element (RRE) and the Rev-RRE complex primarily controls nucleocytoplasmic transport of viral RNAs. Inhibition of Rev-RRE interaction therefore is an attractive target to block viral transport. We have developed a stable cell line carrying a lentiviral vector harboring a rev gene and a co-linear Rev-dependent GFP/luciferase reporter gene cassette and thus constitutively expressing the reporter proteins. Dose-dependent luciferase activity inhibition in the indicator cell line by known small molecule inhibitors Proflavin and K37 established the specificity of the assay. This novel single step assay, that involves use of very small amount of reagents/cells and addition of test material as the only manipulation, can therefore be useful for screening therapeutically potential Rev-RRE interaction inhibitors.